HIF2α activation and mitochondrial deficit due to iron chelation cause retinal atrophy

Abstract Iron accumulation causes cell death and disrupts tissue functions, which necessitates chelation therapy to reduce iron overload. However, clinical utilization of deferoxamine (DFO), an iron chelator, has been documented to give rise to systemic adverse effects, including ocular toxicity. This study provided the pathogenic and molecular basis for DFO‐related retinopathy and identified retinal pigment epithelium (RPE) as the target tissue in DFO‐related retinopathy. Our modeling demonstrated the susceptibility of RPE to DFO compared with the neuroretina. Intriguingly, we established upregulation of hypoxia inducible factor (HIF) 2α and mitochondrial deficit as the most prominent pathogenesis underlying the RPE atrophy. Moreover, suppressing hyperactivity of HIF2α and preserving mitochondrial dysfunction by α‐ketoglutarate (AKG) protects the RPE against lesions both in vitro and in vivo. This supported our observation that AKG supplementation alleviates visual impairment in a patient undergoing DFO‐chelation therapy. Overall, our study established a significant role of iron deficiency in initiating DFO‐related RPE atrophy. Inhibiting HIF2α and rescuing mitochondrial function by AKG protect RPE cells and can potentially ameliorate patients' visual function.

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Lise Roth
Lise Roth, PhD Senior Editor EMBO Molecular Medicine ***** Reviewer's comments ***** Referee #1 (Remarks for Author): Deferoxamine treatment, an iron chelator can have severe adverse effects, including ocular toxicity. The group here presents interesting data from patients, a mouse model and iPS-derived RPE cells. The paper reports a specific susceptibility of RPE cells to deferoxamine that leads to mitochondrial (HIF2a) dysfunction leading then to RPE atrophy.
I am very enthusiastic about the first parts of the paper that show a clear translational approach in patients and mice. The last part with a-ketoglutarate (AKG) as a direct therapy for RPE damage via HIF2A inhibition is less clear. There have been several papers that citric cycle intermediates can alleviate retinal degeneration in more general setting of IRDs.This point should be at least discussed carefully.
Minor points: 1. There is a hypothesis that hyperreflective spots seen in mouse retinal fundus imaging of damage models could potentially represent reactive immune cells (microglia). It would be worth to have a look on retinal and RPE/choroidal flat mounts stained with markers such as Iba1 to rule out this immune-mediated phenomenon in the mouse damage/treatment studies. 2. Fig. 2K-N. The phalloiding staining of the (damaged) RPE cell network should be supplemented with a second verification marker such as zonula occludens protein 1 (ZO1) (as has been done for in vitro RPE in Fig. 3). 3. I wonder whether the strongly induced expression in the iRPE cells in VEGF transcripts really translates to VEGF-A protein expression, which would also indicate a proangiogenic trigger of deferoxamine.
Referee #2 (Comments on Novelty/Model System for Author): The major conclusions need to be confirmed in the mouse model. The iRPE data are of unclear validity due to the extremely high concentration of DFO used.
Referee #2 (Remarks for Author): In this article, the authors address the problem of Deferoxamine (DFO) toxicity on the eye. DFO is an iron chelator in clinical use to remove iron from overloaded patients with thalassemia, especially those dependent on red cell transfusions. Ocular toxicity occurs in less than 10% of patients taking DFO, and it is unclear why they are particularly susceptible and what the mechanism of toxicity is. The manuscript presents clinical data from 4 patients, mouse model experiments and iRPE experiments. The authors come to the surprising conclusion that the toxic effects of DFO are due to iron starvation of the RPE, manifested in particular by mitochondrial respiratory dysfunction and HIF2 alpha induction. The toxic effects could be partially rescued by treatment with alpha keto glutarate, a PHD substrate involved in HIF induction.
Pharmacology -How were the DFO doses used arrived at? The dose for the mice was 100 mg/kg, somewhat higher than the recommended dose for human use, but in the same range. What is the effective drug concentration here? The dose for the iRPE experiments, 100 mM, seems to be a huge excess over the recommended dosing. Toxicity from this high dose may or may not reflect the situation in intact retina in an organism (mouse or human) treated with the recommended DFO dosing. The conclusions regarding the role of mitochondrial respiration and HIF2 alpha induction in DFO toxicity are based on the experiments with iRPE with the extra high DFO concentration. If possible these results should be confirmed in the mouse model. Does the RPE tested in seahorse lose respiratory capacity? Is HIF2 alpha induced in RPE of the mouse treated with DFO. Does AKG rescue in the mouse setting.
A major finding of the paper is that the toxicity of DFO relates to iron starvation. In the write-up on Deferoxamine (Drug information, Up-to-Date), it says "Ocular disturbances ...have been reported following prolonged administration at high doses or in patients with low ferritin levels..." The database goes on to say, "a lower dose may be required if ferritin levels are low. In general the therapeutic index should be kept at <0.05. Therapeutic index = mean daily deferoxamine dose (mg/kg)/ferritin (mcg/L)." The implication of this recommendation is that DFO toxicity may occur if the patient is already iron starved (has low ferritin level). This is consistent with the conclusions of the manuscript that DFO toxicity is mediated by iron starvation. Suggest a) the authors should discuss this clinical recommendation and how it accords with the conclusions of their paper, b) the authors should go back into the clinical histories of their 4 chelated thalassemic patients to see if perhaps they were chelated in a setting in which ferritin was already low.
The data are clear, the paper is well written, and the paper is thoroughly referenced. The conclusions are important and interesting.
Referee #3 (Comments on Novelty/Model System for Author): Please see "comments to the Authors": certain points of the protocol might be presented and further explained in the main text to facilitate the reading and understanding of the experimental sets; some questions about the clinical part of the study also need clarification.
Referee #3 (Remarks for Author): The study (manuscript entitled "HIF2α Activation and Mitochondrial Deficit due to Iron Chelation Cause Retinal Atrophy") is based on the documentation of ocular toxicity associated with the clinical use of the iron chelator deferoxamine (DFO). The scientific question is of scientific and clinical interest considering the dual role of iron in the ocular health, namely on the one hand the iron deficiency in the eye can significantly damage the metabolic homeostasis of the retinal pigment epithelial (RPE) and, on the other hand the intracellular accumulation of iron was associated with harmful effects, e.g. oxidative stress, increased levels of pro-inflammatory cholesterol in retina, etc.
Here, the Authors aimed to uncover a target mechanism as a therapeutic solution to DFO-related retinopathy. By suppressing hyper-activity of HIF2α and preserving mitochondrial dysfunction by α-ketoglutarate (AKG) it was found that i) AKG protects the RPE against lesions both in vitro and in vivo; ii) demonstrated a susceptibility of RPE to DFO (compared to neuroretina) and iii) identified upregulation of hypoxia inducible factor (HIF) 2a -a master regulator of oxygen homeostasis and aerobic glycolysis implicated in angiogenesis, extracellular matrix dynamics and cell survival-and mitochondrial deficit as the most prominent pathogenesis underlying the DFO RPE atrophy. Since the AKG supplementation was found to prevent RPE cell death and alleviate patient visual decline due to DFO intake, it was suggested as a possible therapeutic agent against DFO-associated retinopathy.
Comments, questions, suggestions: "Four patients of ß-thalassemia intermedia with a history of blood transfusion were subject to iron chelation by DFO for at least 16 years": Please specify the retrospective character of the study. The Case IV only had a history of taking AKG (2g/day) as a supplement for 18 months? How this case could be compared to the other three cases (patients who did not receive AKG)? How the protective effect of AKG in this clinical case could specifically attributed to AKG? From only one clinical observation/documentation, could we be sure that the secondary photoreceptor malfunction or degeneration was halted exclusively by AKG? The demographic characteristics of the 4 patients are very different, specifically Case II is only 26 yrs old (though with 17 yrs history of chelation therapy), while Case IV is 64 yrs old (16 yrs history of chelation therapy): could you comment your results considering that iron levels in the retina have been reported increasing with age in human eyes?
"We found a progression of the spotting presentation throughout the course of DFO injection in mice (Figure 2A -C)." The time points could be mentioned also in the main text. Otherwise, the reader has to check the "Material and Method" section to find for "...100 mg/kg DFO three times a week from the weaning age onwards" that "mice were injected with DFO for 10 months...". But then (page 9) we see that "In addition to anatomical changes, we characterized functional impairment of both neuroretina and RPE by ERG on mice injected with DFO for six months." or "mice treated with DFO for five and ten months." Please make clear the time-schedule to avoid confusion.
Some wording modifications could also make the text more readily accessible, e.g. "In order to specify a potential impact of RPE pathology in response to DFO toxicity" may be changed here and in other phrases to "... in response to DFO".
Can you comment the significance/clinical relevance of the findings about "toxic effect of DFO at 10 mM" at different time intervals post treatment?
Regarding the impact of the HIFα pathway on RPE pathology sounds and the comparison between HIF1 and HIF2, please comment other possible mechanisms, if any.
Since AKG was previously reported to halt the progression of retinal degeneration in mice (as mentioned by the Authors: Rowe et al, 2021), please underline the input of your finding to the field.
The discussion (page 19) states that RPE abnormalities have previously been characterized and postulated as primary lesions associated with DFO chelation therapy. Maybe the here-reported results should be emphasized in a comparative manner in order to highlight their novelty.
The potential of transferrin for treatment of retinal pathologies (compared to other chelators or antioxidants) could be mentioned. It would be of interest to mention (introduction or discussion) if other iron chelators provoke retinal toxicity. The Authors should better position their work versus the Dunaieff or Courtois/Behar-Cohen works.

Dear Editor and Reviewers,
We are pleased with the positive response to our manuscript titled, "HIF2α Activation and Mitochondrial Deficit due to Iron Chelation Cause Retinal Atrophy" for consideration in EMBO Molecular Medicine, and excited by the degree of the reviewers' enthusiasm for our work. We are also grateful for all of the thoughtful and constructive comments.
This letter provides point-by-point responses to the Reviewers' comments. The Reviewers' concerns, shown in regular type, are followed by our response that are underlined: We hope you will find out responses adequately address all the reviewers' comments and that our submission is acceptable for publication in EMBO Molecular Medicine.
Thank you for your consideration.

Referee #1 (Remarks for Author):
Deferoxamine treatment, an iron chelator can have severe adverse effects, including ocular toxicity. The group here presents interesting data from patients, a mouse model and iPS-derived RPE cells. The paper reports a specific susceptibility of RPE cells to deferoxamine that leads to mitochondrial (HIF2a) dysfunction leading then to RPE atrophy.
I am very enthusiastic about the first parts of the paper that show a clear translational approach in patients and mice. The last part with a-ketoglutarate (AKG) as a direct therapy for RPE damage via HIF2A inhibition is less clear. There have been several papers that citric cycle intermediates can alleviate retinal degeneration in more general setting of IRDs. This point should be at least discussed carefully.

A:
We understand the reviewer's concern and appreciate the suggestions. We provided further characterization of the inhibitory effect of AKG on HIF1α and 2α, including immunoblotting analyses on the RPE from the mice that were subject to chronic treatment of DFO with and without AKG treatment. The data are presented in Figure EV 4 and Figure 7. Additionally, we cited and discussed the rescuing effect of intermediary metabolites identified previously, which focused on initial screening schemes to identify protective agents and highlighted phenotypic modification (Paragraph 2, Page 17). Phenotypic protection was reported in the neural system (1-3), especially in the context of inherited disorders. This study further validated the rescue effects of AKG in an acquired degenerative disease induced by drug 5th Nov 2022 1st Authors' Response to Reviewers toxicity, which could be more prevalent in the clinic. Additionally, we highlighted the molecular basis that underpins this protective effect and raised the concern about potential limitations in applying intermediary metabolites for protecting against neurodegeneration.
Minor points: 1. There is a hypothesis that hyperreflective spots seen in mouse retinal fundus imaging of damage models could potentially represent reactive immune cells (microglia). It would be worth to have a look on retinal and RPE/choroidal flat mounts stained with markers such as Iba1 to rule out this immune-mediated phenomenon in the mouse damage/treatment studies. A: We tested for the presence of microglia in RPE flat mount harvested from the mice that received DFO treatment for 10 months. The age-matched untreated mice were used as the control. Based on our results, IBA1-positive cells were scarcely present in the DFO-treated mice, which is comparable to the untreated controls. The data have been included in Appendix Figure S1A -C. 2. Fig. 2K-N. The phalloidin staining of the (damaged) RPE cell network should be supplemented with a second verification marker such as zonula occludens protein 1 (ZO1) (as has been done for in vitro RPE in Fig. 3). A: As the reviewer advised, we included the IHC for ZO1 in RPE flat mounts collected from the mice with more than 10 months of DFO treatment. DFO-treated RPE appeared polymorphous and lost the hexagonal packing compared with the untreated controls. Please see Appendix Figure S1D and E. 3. I wonder whether the strongly induced expression in the iRPE cells in VEGF transcripts really translates to VEGF-A protein expression, which would also indicate a proangiogenic trigger of deferoxamine. A: Thanks for the intriguing question. We measured the secretion of VEGF-A in the DFO-treated iRPE cells. As anticipated, an elevation of secreted VEGF-A was present but not significant, 24 hours post-DFO treatment. Whereas a significant increase in VEGF-A was detected in the iRPE cells with DFO treatment for 48 hours. In fact, increased VEGF-A due to DFO stimulation was previously reported (4). Please find the analyses added in Appendix Figure S1F and G.

Referee #2 (Comments on Novelty/Model System for Author):
The major conclusions need to be confirmed in the mouse model. The iRPE data are of unclear validity due to the extremely high concentration of DFO used.

A:
We are grateful for the reviewer's recommendation and we validated phenotypic changes associated with DFO toxic effect in the mice (Figures 2 and 6). As a major finding, we tested HIFα levels in mouse RPE, which corroborated our in-vitro observations. The data are presented in Figure 7H and Figure EV 4F.
Admittedly, recapitulating phenotypic changes with mouse RPE, especially at the molecular level is challenging. Several aspects of modeling and technical difficulties hinder us from making direct interpretations, including: 1. Unlike genetically engineered models that are easier to yield consistent outcomes, tissues collected from pharmacological-induced mouse models via systemic delivery are volatile and lack biochemical stability for post-mortem examinations, which is exemplified by the abundant degradation of HIFα tested in our mouse RPE. 2. Isolation of RPE from the back of the eye allows for contamination by other types of retinal cells, as well as the choroid. These interfere with the outcomes, especially in consideration of making claims about different metabolic systems in the neuroretina. 3. We attempted to delineate the metabolic profiles of the RPE under the influence of DFO as we identified in vitro. Monolayered RPE, harvested from the mice treated by DFO with or without AKG, was used for the Extracellular Flux Seahorse assay by following the protocol for testing mitochondrial functions with frozen tissues. However, our results failed to reveal statistical significance among the groups of untreated, DFO treated and DFO treated with AKG supplementation due to variation. It should be noted that seahorse is a less sensitive approach to capturing delicate changes in the animal tissue. Handling of tissue dissection and availability of frozen tissues could be even less sufficient to detect transient and subtle alterations in mitochondria as well as their interplay with aerobic glycolysis.
This test has been completed. We are willing to share it upon the reviewers' request.
Referee #2 (Remarks for Author): In this article, the authors address the problem of Deferoxamine (DFO) toxicity on the eye. DFO is an iron chelator in clinical use to remove iron from overloaded patients with thalassemia, especially those dependent on red cell transfusions. Ocular toxicity occurs in less than 10% of patients taking DFO, and it is unclear why they are particularly susceptible and what the mechanism of toxicity is. The manuscript presents clinical data from 4 patients, mouse model experiments and iRPE experiments. The authors come to the surprising conclusion that the toxic effects of DFO are due to iron starvation of the RPE, manifested in particular by mitochondrial respiratory dysfunction and HIF2 alpha induction. The toxic effects could be partially rescued by treatment with alpha keto glutarate, a PHD substrate involved in HIF induction.
Pharmacology -How were the DFO doses used arrived at? The dose for the mice was 100 mg/kg, somewhat higher than the recommended dose for human use, but in the same range. What is the effective drug concentration here? The dose for the iRPE experiments, 100 mM, seems to be a huge excess over the recommended dosing. Toxicity from this high dose may or may not reflect the situation in intact retina in an organism (mouse or human) treated with the recommended DFO dosing. The conclusions regarding the role of mitochondrial respiration and HIF2 alpha induction in DFO toxicity are based on the experiments with iRPE with the extra high DFO concentration. If possible these results should be confirmed in the mouse model. Does the RPE tested in seahorse lose respiratory capacity? Is HIF2 alpha induced in RPE of the mouse treated with DFO. Does AKG rescue in the mouse setting.
A: Indeed, the concentration used in this study, especially the one for in-vitro assays is much higher than is frequently used in other research studies. We chose this high dosage as we desired to accelerate phenotypic manifestations in iRPE, since DFO toxicity takes more than 10 years to develop in human patients. We included this information in the manuscript (Paragraph 2, Page 9). For choosing the dosage in the mice, we referred to the clinical dosage for inducing toxicity in order to simulate the progression of the phenotypes in the most efficient manner. Meanwhile subtle or transient changes can be captured within a reasonable duration of time.
The in-vivo phenotyping of DFO and AKG-treated mice was included in Figure 2 and Figure 6. At the molecular level, we included immunoblotting against HIFα in mouse RPE ( Figure 7H) as further evidence of hyperactivation of HIF2α by DFO, which can be suppressed by AKG. As aforementioned, we attempted to recapitulate the suppressive effects of DFO on mitochondrial respiration using the seahorse assay. The results lack significant differences. This is likely due to the fact that some mice remained robustly resistant to the toxic effect even at a high dosage, similar to the variation observed in human patients taking DFO We touched upon part to the manuscript (Paragraph 2, Page 19).
A large proportion of molecular changes in vitro take place at a 24-hour time point while gross phenotypic changes were hardly seen at this time. The 48-hour time point appears to be more compelling in exhibiting phenotypes. Additionally, we were aware that DFO is conventionally regarded as being pro-survival in cells undergoing ferroptosis. However, high-dose DFO, despite being less investigated, has been reported to induce the death of cancer cells, which corroborates our findings. Additionally, the guideline, based on UpToDate, documented such toxicity at a high dose as well based on the reviewer's feedback, which further supports our findings in this study. We have revised the text and addressed the differential impact of DFO at different doses in the discussion section (Paragraph 1, Page 16).
A major finding of the paper is that the toxicity of DFO relates to iron starvation. In the write-up on Deferoxamine (Drug information, Up-to-Date), it says "Ocular disturbances ...have been reported following prolonged administration at high doses or in patients with low ferritin levels..." The database goes on to say, "a lower dose may be required if ferritin levels are low. In general, the therapeutic index should be kept at <0.05. Therapeutic index = mean daily deferoxamine dose (mg/kg)/ferritin (mcg/L)." The implication of this recommendation is that DFO toxicity may occur if the patient is already iron starved (has low ferritin level). This is consistent with the conclusions of the manuscript that DFO toxicity is mediated by iron starvation. Suggest a) the authors should discuss this clinical recommendation and how it accords with the conclusions of their paper, b) the authors should go back into the clinical histories of their 4 chelated thalassemic patients to see if perhaps they were chelated in a setting in which ferritin was already low.

A:
We appreciated the reviewer's comment about clinical guidance for the precautions of the usage of DFO. We revisited our patients' blood test results. By referring to relevant publications and consulting hematologists, we found that serum ferritin higher than the normal range is common in transfusion-dependent patients. Typically, physicians are less likely to to reduce the elevated level of serum ferritin to the normal range for these patients in the clinic. Intriguingly, we learned that ferritin assays lack specificity and sensitivity, being subject to systemic conditions such as inflammatory reactions, etc. Reliable examinations, including MRI and invasive tissue biopsy, need to be considered if local iron overload is highly suspected. We have addressed this in the discussion section (Paragraph 2, Page 19) and included patients' ferritin levels over the years in Appendix Table S1.
The data are clear, the paper is well written, and the paper is thoroughly referenced. The conclusions are important and interesting.

Referee #3 (Comments on Novelty/Model System for Author):
Please see "comments to the Authors": certain points of the protocol might be presented and further explained in the main text to facilitate the reading and understanding of the experimental sets; some questions about the clinical part of the study also need clarification.
Referee #3 (Remarks for Author): The study (manuscript entitled "HIF2α Activation and Mitochondrial Deficit due to Iron Chelation Cause Retinal Atrophy") is based on the documentation of ocular toxicity associated with the clinical use of the iron chelator deferoxamine (DFO). The scientific question is of scientific and clinical interest considering the dual role of iron in the ocular health, namely on the one hand the iron deficiency in the eye can significantly damage the metabolic homeostasis of the retinal pigment epithelial (RPE) and, on the other hand the intracellular accumulation of iron was associated with harmful effects, e.g. oxidative stress, increased levels of pro-inflammatory cholesterol in retina, etc.
Here, the Authors aimed to uncover a target mechanism as a therapeutic solution to DFO-related retinopathy. By suppressing hyper-activity of HIF2α and preserving mitochondrial dysfunction by α-ketoglutarate (AKG) it was found that i) AKG protects the RPE against lesions both in vitro and in vivo; ii) demonstrated a susceptibility of RPE to DFO (compared to neuroretina) and iii) identified upregulation of hypoxia inducible factor (HIF) 2a -a master regulator of oxygen homeostasis and aerobic glycolysis implicated in angiogenesis, extracellular matrix dynamics and cell survival-and mitochondrial deficit as the most prominent pathogenesis underlying the DFO RPE atrophy. Since the AKG supplementation was found to prevent RPE cell death and alleviate patient visual decline due to DFO intake, it was suggested as a possible therapeutic agent against DFO-associated retinopathy.
Comments, questions, suggestions: "Four patients of ß-thalassemia intermedia with a history of blood transfusion were subject to iron chelation by DFO for at least 16 years": Please specify the retrospective character of the study.  1, Page 7). Furthermore, we would like to clarify that potential amelioration in Case IV was compared to himself before intake of AKG only, which is an observation in a longitudinal manner, and we made sure not to overstate the claim on whether the protection is a direct result of AKG supplementation. Admittedly, the cohort overall is small. Patients at the stage of ophthalmic presentations in the clinic are rare. Therefore, it is difficult to identify a large number of patients with orphan disorders to conduct a systematic clinical review, which sufficiently highlights the significance of our work by using in vitro and in vivo models.
We have noted this limitation in the discussion section (Paragraph 2, Page 19). Excitingly, we observed protection against morphological damage due to DFO toxicity as well as mitigation of neuroretinal degeneration by AKG.
In terms of the fluctuation of iron in the retina, we also read the report about the accumulation of iron with age in the human population. However, the ophthalmic presentations in the patients in this study lack common and classic signs of iron accumulation. We have elucidated this regard in both the result and discussion sections as the reviewer recommended (Paragraph 1, Page 5; Paragraph 1, Page 16).
"We found a progression of the spotting presentation throughout the course of DFO injection in mice (Figure 2A -C)." The time points could be mentioned also in the main text. Otherwise, the reader has to check the "Material and Method" section to find for "...100 mg/kg DFO three times a week from the weaning age onwards" that "mice were injected with DFO for 10 months...". But then (page 9) we see that "In addition to anatomical changes, we characterized functional impairment of both neuroretina and RPE by ERG on mice injected with DFO for six months." or "mice treated with DFO for five and ten months." Please make clear the time-schedule to avoid confusion.

A:
We thank the reviewer for the meticulous comments and have clarified the timeline throughout the manuscript. The regimen for DFO administration is mentioned in the results section. We Some wording modifications could also make the text more readily accessible, e.g. "In order to specify a potential impact of RPE pathology in response to DFO toxicity" may be changed here and in other phrases to "... in response to DFO".'

A:
We appreciate the reviewer's suggestion and have modified the wording accordingly to minimize partial interpretations of the results.
Can you comment the significance/clinical relevance of the findings about "toxic effect of DFO at 10 (100?) mM" at different time intervals post treatment?
A: We thank the reviewer for raising this point. In fact, we detected the in vitro and in vivo manifestations linked to DFO's toxic effects at different time points. Fundamental molecular changes tend to be initiated at an earlier time point while gross phenotypical changes are not manifested until a later time point, which implies a drastic disruption has taken place long before clinical manifestations. This reflects the patients' conditions, where DFO toxicity is a prolonged and accumulative process. Additionally, we found that the rescuing effects could be optimized if AKG was applied at an earlier time point. AKG is insufficient to reverse the course of DFOrelated damage as the toxic effects accumulate over time. Clinically, we believe that DFO toxicity should be monitored regularly at an early stage even before the appearance of pathological hyper-fluorescent lesions. Therapeutic interventions should be employed at an early stage or even as a preventive measure. We have noted it in the discussion section (Paragraph 2, Page 17; Paragraph 2, Page 19).
Regarding the impact of the HIFα pathway on RPE pathology sounds and the comparison between HIF1 and HIF2, please comment other possible mechanisms, if any.

A:
The core concept of this study is the perturbation of iron homeostasis due to DFO chelation. Any iron-dependent biochemical reactions or enzymatic activities could be impacted, such as dysfunctional cytochrome c, production of oxidative stress and its sources, etc. Notably, HIFα serves as a master regulator in the majority of these changes.
We have added information about these potential mechanistic explanations for the RPE damage associated with DFO toxicity in the discussion section (Paragraph 2, Page 17).
Since AKG was previously reported to halt the progression of retinal degeneration in mice (as mentioned by the Authors: Rowe et al, 2021), please underline the input of your finding to the field.

A:
We thank the reviewer for the suggestion, and have further highlighted the significance of our study in relation to the previous investigations (Paragraph 1, Page 16; Paragraph 2, Page 17).
The discussion (page 19) states that RPE abnormalities have previously been characterized and postulated as primary lesions associated with DFO chelation therapy. Maybe the here-reported results should be emphasized in a comparative manner to highlight their novelty.
A: As the reviewer recommended, the significance of this study was highlighted as a mechanistic investigation of DFO-related retinopathy with clinic relevance. More importantly, our finding pinpointed HIF2α as a key player and characterized the metabolic profile involved in the RPE pathology that could be extrapolated to a greater scope of neurodegenerative disorders, such as age-related macular degeneration.
The potential of transferrin for the treatment of retinal pathologies (compared to other chelators or antioxidants) could be mentioned. It would be of interest to mention (introduction or discussion) if other iron chelators provoke retinal toxicity. The Authors should better position their work versus the Dunaieff or Courtois/Behar-Cohen works.
A: Based on the reviewer's comments, we carefully addressed similarities and differences between DFO from its counterparts, such as DFP in biological properties and pharmacological toxicity. (Paragraph 1, Pages 16; Paragraph 2, Page 17). Additionally, we plan to conduct further experiments to explore this question in future studies. Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now received feedback from the three referees who originally reviewed your manuscript. As you will see below, they are now supportive of publication, and I am therefore pleased to inform you that we will be able to accept your manuscript once the following editorial points will be addressed: -Acknowledgements: Please make sure that the funding information provided in the manuscript is the same as what is provided in the submission system. -Author contributions: CRediT has replaced the traditional author contributions section because it offers a systematic machinereadable author contributions format that allows for more effective research assessment. Please remove the Authors Contributions from the manuscript and use the free text boxes beneath each contributing author's name in our system to add specific details on the author's contribution. More information is available in our guide to authors. -Please remove the Appendix legends from the main manuscript file.

2/ Figures and Appendix:
- Tables EV1 and EV2 should be renamed Table 1 and 2, or uploaded as separate files.
-The appendix figure and table should be merged in one PDF file with a table of content. Legends should be added. The nomenclature should be corrected to Appendix Figure S1 and Appendix Table S1. 4/ Please provide "The paper explained": EMBO Molecular Medicine articles are accompanied by a summary of the articles to emphasize the major findings in the paper and their medical implications for the non-specialist reader. Please provide a draft summary of your article highlighting -the medical issue you are addressing, -the results obtained and -their clinical impact. This may be edited to ensure that readers understand the significance and context of the research. Please refer to any of our published articles for an example. 5/ Synopsis: Thank you for providing a nice synopsis image. I resized it to fit our format, please let me know if you approve the final image (attached). I also slightly edited the text, please let me know if you agree with the following or amend as you see fit: Deferoxamine (DFO) chelation therapy stabilizes HIF2α and disrupts mitochondrial oxidative phosphorylation, leading to RPE atrophy. Ablating HIF2α and preserving mitochondrial oxidative phosphorylation by α-ketoglutarate (AKG) mitigates RPE cell death, which ameliorates visual impairment in the clinic.
-DFO primarily acts on RPE to disrupt iron homeostasis causing atrophic lesions.
-DFO upregulates HIF2α and undermines mitochondrial oxidative phosphorylation in the RPE, accounting for its susceptibility to iron removal.
-RPE intolerance to HIF2α-induced anaerobic glycolysis contributes to susceptibility to DFO toxicity. RPE survival is sustained by mitochondrial oxidative phosphorylation.
-AKG, an intermediary metabolite of the Krebs cycle, destabilizes HIF2α and preserves mitochondrial respiration capacity, which protects RPE from damage and mitigates visual impairment in the clinic. 6/ As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication.
Please note that the Authors checklist will be published at the end of the RPF.
I look forward to receiving your revised manuscript. The quality of the revised manuscript appears significantly improved, the additional references are relevant, the clinical/translational potential of the findings is well-defined.

10th Dec 2022 2nd Authors' Response to Reviewers
The authors addressed the minor editorial issues. Thank you for providing the revised files. I am pleased to inform you that your manuscript is now accepted for publication in EMBO Molecular Medicine and will be sent to our publisher to be included in the next available issue.
Please note that the bullet points from the synopsis must not exceed 30 words each, I therefore had to slightly modify your synopsis. Please inform us immediately if you do not agree with the following: Deferoxamine (DFO) stabilizes HIF2α and disrupts mitochondrial oxidative phosphorylation, leading to RPE atrophy. Inhibiting HIF2α and preserving mitochondrial oxidative phosphorylation by α-ketoglutarate (AKG) mitigates RPE cell death, which ameliorates visual impairment in the clinic.
-DFO primarily acts on RPE and disrupts its iron homeostasis, causing atrophic lesions.
-DFO upregulates HIF2α and undermines mitochondrial oxidative phosphorylation in the RPE, accounting for its susceptibility to iron depletion.
-RPE intolerance to HIF2α-induced anaerobic glycolysis contributes to susceptibility to DFO toxicity.
-RPE survival is sustained by mitochondrial OXPHOS, whereas the anaerobic glycolytic photoreceptor is initially spared from enhanced HIF levels.
-AKG, an intermediary metabolite of the Krebs cycle, destabilizes HIF2α and preserves mitochondrial respiration capacity, which protects RPE from damage and mitigates visual impairment in the clinic.
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If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.
Yes Table EV1 Core facilities Information included in the manuscript?
In which section is the information available?
(Reagents and Tools the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.

Reporting Checklist for Life Science Articles (updated January 2022)
ideally, figure panels should include only measurements that are directly comparable to each other and obtained with the same assay. plots include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates.
the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
a statement of how many times the experiment shown was independently replicated in the laboratory.
-common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; Please complete ALL of the questions below. Select "Not Applicable" only when the requested information is not relevant for your study.
if n<5, the individual data points from each experiment should be plotted. Any statistical test employed should be justified. Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Presentation.
Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name). the assay(s) and method(s) used to carry out the reported observations and measurements. an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.

Study protocol
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Include a statement about sample size estimate even if no statistical methods were used. Yes Figure legends Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, have they been described?

Not Applicable
Include a statement about blinding even if no blinding was done. Not Applicable Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre-established?
If sample or data points were omitted from analysis, report if this was due to attrition or intentional exclusion and provide justification.

Methods and Materials
For every figure, are statistical tests justified as appropriate? Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Is there an estimate of variation within each group of data? Is the variance similar between the groups that are being statistically compared?

Sample definition and in-laboratory replication Information included in the manuscript?
In which section is the information available?
(Reagents and Tools In the figure legends: state number of times the experiment was replicated in laboratory. Yes Figure Legends In the figure legends: define whether data describe technical or biological replicates. Yes Figure Legends, Methods and Materials